This proposal concerns the principal mechanisms of protein degradation in the cell cycle. These mechanisms are central to our understanding of the basic workings of the cell cycle machinery in all eukaryotic cells and may be of particular importance in checkpoint controls that are thought to be defective in cancer. We are proposing to extend the biochemical approach using assays in frog egg extracts and purified biochemical activities to address the following issues. 1) The structure and function of the anaphase promoting complex (APC), an assemblage of 8 proteins that mediates exit from mitosis by catalyzing the transfer of ubiquitin to cyclin and other substrates. We propose to complete our characterization of its structure to ask how it carries out ubiquitin conjugation to cyclin and other substrates. 2)The mechnism of activation of APC in mitosis and its inactivation in G1. We wish to understand the intracellular signals regulating APC activity. 3) Checkpoint controls of APC in cytostatic factor arrest in meiotic metaphase and spindle damage control in cultured cells. We propose to purify and characterize inhibitory activities that regulate APC from signals extrinsic to the cell cycle. 4) Identification of substrates containing the destruction box. Using small pool screening we plan to identify potential M-phase proteolytic substrates and to characterize a novel D-box containing protein that may be involved in chromosome assembly and DNA replication. 5) Understanding the proteolytic steps in initiation of DNA replication. Following experiments on the vertebrate homolog of cdc34, we plan to characterize its pathway of ubiquitin mediated degradation and potential substrates relative to the onset of DNA replication.